Epidemics and pandemics caused by viral diseases are still claiming human lives and are impacting global economy. Influenza is responsible for millions of lost work days and visits to the doctor, hundreds of thousands of hospitalizations worldwide (Couch 1993, Ann. NY. Acad. Sci 685; 803,), tens of thousands of excess deaths (Collins & Lehmann 1953 Public Health Monographs 213:1; Glezen 1982 Am. J. Public Health 77:712) and billions of Euros in terms of health-care costs (Williams et al. 1988, Ann. Intern. Med. 108:616). Both influenza A and B viruses have in the past been responsible for these epidemics in humans, thus besides influenza A also influenza B virus surface antigens are an essential component of any vaccine effective in reducing influenza morbidity. Influenza viruses belong to the Orthomyxoviridae family and are characterized by segmental negative-strand RNA genomes that add up to total sizes of 13.6 to 14.6 kb, respectively. Genomic viral RNA must be packaged into viral particles in order for the virus to be transmitted. The process by which progeny viral particles are assembled and the protein/protein interactions occur during assembly are similar within RNA viruses. The formation of virus particles ensures the efficient transmission of the RNA genome from one host cell to another within a single host or among different host organisms. The influenza virions consist of an internal ribonucleoprotein core (a helical nucleocapsid) containing the single-stranded RNA genome, and an outer lipoprotein envelope lined inside by a matrix protein (M1). The segmented genome of influenza A virus consists of eight molecules of linear, single-stranded RNAs of negative polarity, which encodes eleven (some influenza A strains ten) polypeptides, including: the RNA-dependent RNA polymerase proteins (PB2, PB1 and PA) and nucleoprotein (NP) which form the nucleocapsid; the matrix membrane proteins (M1, M2 or BM2); two surface glycoproteins which project from the lipid containing envelope: hemagglutinin (HA) and neuraminidase (NA); the nonstructural protein (NS1) and nuclear export protein (NEP). Most influenza A strains also encode an eleventh protein (PB1-F2) believed to have proapoptotic properties, whereas only influenza B viruses express the NB protein that might contribute to viral virulence (Hatta and Kawaoka, 2003, J. Virol., 77, 6050-6054). There are further minor differences between influenza A and B viruses in their expression strategies of gene products encoded by the viral NA and M gene segments (Lamb and Horvath, 1991, Trends Genet. 7:261-266). Significant biological and epidemiological differences are indicated by the almost exclusive confinement of influenza B viruses to humans, although there have already been studies isolating influenza B virus from seals indicating that there might also be a bigger reservoir of different organisms. Influenza A viruses have a very broad reservoir in many avian and mammalian species.
Transcription and replication of the genome takes place in the nucleus and assembly occurs via budding from the plasma membrane. The viruses can reassort genes during mixed infections. Influenza virus adsorbs via HA to sialyloligosaccharides in cell membrane glycoproteins and glycolipids. Following endocytosis of the virion, a conformational change in the HA molecule occurs within the cellular endosome which facilitates membrane fusion, thus triggering uncoating. The nucleocapsid migrates to the nucleus where viral mRNA is transcribed. Viral mRNA is transcribed by a unique mechanism in which viral endonuclease cleaves the capped 5′-terminus from cellular heterologous mRNAs which then serve as primers for transcription of viral RNA templates by the viral transcriptase. Transcripts terminate at sites 15 to 22 bases from the ends of their templates, where oligo(U) sequences act as signals for the addition of poly(A) tracts. Of the eight viral RNA molecules so produced during influenza A replication, six are monocistronic messages that are translated directly into the proteins representing HA, NA, NP and the viral polymerase proteins, PB2, PB1 and PA. The other two transcripts undergo splicing, each yielding two mRNAs which are translated in different reading frames to produce M1, M2, NS1 and NEP. In other words, the eight viral RNA segments code for eleven proteins: nine structural and 2 nonstructural (NS1 and the recently identified PB1-F2) proteins. Influenza B uses a different coding strategy for 2 proteins, namely NB and BM2. The former is translated from an overlapping reading frame of the NA gene and the latter is expressed via an overlapping stop-start codon from the M gene.
Vaccination is presently seen as the best way to protect humans against influenza. When healthy adults get immunized, currently available vaccines prevent clinical disease in 70-90% of cases. This level is reduced to 30-70% in those over the age of 65 and drops still further in those over 65 living in nursing homes (Strategic Perspective 2001: The Antiviral Market. Datamonitor. p. 59). The virus's frequent antigenic changes further contribute to a large death toll because not even annual vaccination can guarantee protection.
Vaccination is accomplished with commercially available, chemically inactivated (killed) or live attenuated influenza virus vaccines. Unfortunately, inactivated vaccines can hardly induce cross-protective immunity and therefore the vaccine strain must exactly fit to the antigenic properties of the future unknown pandemic strain.
Replication deficient influenza A viruses are supposed to overcome the safety issues in view of viral shedding. These can be influenza A mutants having deletions of the NS1 protein. The absence of the NS1 protein renders this virus replication-deficient in the respiratory tract of vaccinated mammalians. Upon intranasal administration, the vaccine virus is able to initiate abortive infection in mucosal tissues, without the effect of viral shedding. At the same time the virus stimulates local cytokine response and evokes a B- and T-cell mediated protective immune response.
Influenza B viruses mostly require laborious and time consuming adaptation to reach sufficient growth on Vero cells. Influenza B NS1 mutants which are able to replicate to high titres on Vero cells in addition to an interferon sensitive phenotype due to the abrogated function of NS1 are not described in the literature. Currently, the only published influenza B virus completely lacking the NS1 ORF does not replicate efficiently in Vero cells (titres of 1.7-2.5*102 FFU/ml using an moi of 0.1 and no detectable titres at moi of 0,001, respectively. Dauber et. al; Journal of Virology, February 2004, p. 1865-1872). An influenza B NS1 deletion mutant consisting of the amino-terminal 16 aa is also highly attenuated in replication with maximum titres of approx. 104 FFU/ml. (Hai et. Al; Journal of Virology; November 2008, p. 10580-90
Reflecting the need to develop vaccine formulations of high safety containing influenza B antigenic compounds, there is a great demand in developing influenza B strains which are attenuated due to the abrogated function of NS1 but still show high growth properties in cell culture.